Characterizing the portability of phage-encoded homologous recombination proteins

Bacteriophage single-stranded DNA annealing proteins (SSAPs) interact with the C termini of single-stranded binding proteins in host bacteria, a finding that enables engineering of enhanced SSAP portability and DNA recombineering activities. Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded single-stranded DNA annealing proteins (SSAPs) improve HR 1,000-fold above endogenous levels. However, they are not broadly functional. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus and Caulobacter crescentus, we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host's single-stranded DNA-binding protein (SSB) and are portable between species only if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with SSBs can significantly improve genome editing efficiency, in some species enabling SSAP functionality even without host compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes that are inaccessible through sequential nucleotide conversions.

Automated summary

Bacteriophage single-stranded DNA annealing proteins (SSAPs) interact with the C termini of single-stranded binding proteins in host bacteria, enhancing genome editing efficiency. Co-expressing SSAPs with SSBs can improve editing efficiency, and SSAPs can be transferred between species only if compatibility with the host domain is maintained. High-efficiency HR surpasses mutational capacity of common random mutagenesis methods, generating exceptional phenotypes.